The goal of this proposal is to develop an in vitro test to improve selection of therapies for individual breast cancer patients be predicting response to methotrexate. Existing assays for breast cancer cells cannot determine methotrexate sensitivity because of requirements for feeder layers and medium components which can rescue cells. This proposal will evaluate two different approaches: (1) a clonogenic assay in which a serum-free medium based upon MCDB 170 is modified so that methotrexate toxicity is achieved, and (2) a new assay for methotrexate which measures uptake of fluorescein-conjugated drug using a fluorescence activated cell sorter (FACS) and which does not require clonal growth. Both of these assays were developed in Phase I and will be compared in this Phase II, project for reliability and practicality for commercialization. In additional studies, mechanisms of resistance to methotrexate will be evaluated and normal breast epithelial cells will be compared with breast cancer cells to gain insight on changes which occur during the neoplastic process. In summary, the proposed assays may be useful for predictive patient sensitivity testing and may provide a basis for improved drug design.